The invention concerns a method for imaging, measuring, and interacting with microscopic three-dimensional structures. The invention further concerns an arrangement for imaging and measuring microscopic three-dimensional structures.
As a user works at the microscope, image details (differing according to the application) are always in his or her field of view. In all microscopic applications, for example, there exists a class of functions for measuring morphological and densitometric properties. Morphology analyzes conformational and geometrical features, and densitometry analyzes intensity distribution features; in practice, mixed forms may often be encountered. This class of functions also constitutes the basis for many automatic adjustment operations. In present-day systems, the user works with image details by marking them on the screen with a suitable graphical mechanism (thus defining a geometry) and selecting a desired function of the system, the sequence described here being arbitrary. For three-dimensional geometries, this is a difficult process.
Three-dimensional geometric structures within three-dimensional image stacks that have been generated from data imaged with a microscope are difficult to apprehend mentally. In the real world, many users have only a partial three-dimensional conceptual capability, since 3D models demand a complex mental construction process and thus a certain amount of practice. This is particularly difficult when working with the raw data of a 3D microscope in the form of sectional images, in which case the user must completely reconstruct the three-dimensionality mentally. Measurement obliquely in space of continuous lengths that are not acquired in one section requires, on the basis of the acquired sections, complex navigation through the stack of acquired two-dimensional image data. If the user has no ability to conceptualize in three dimensions, the structure of interest can be identified only with considerable effort.
Concrete examples from microscopy of actions in whose context this problem arises include:
a)Statistical analysis of local properties of images and volumetric image stacks (profiles, histograms, co-localizations, material roughness);
b)Observation of physiological reactions in living cells and in individual compartments (parts of a cell distinguishable in terms of metabolism or structure) thereof;
c)Zoom operations;
d)Aligning the image field;
e)Controlling actuators;
f)Defining locally different excitation and detection parameters;
g)Automated control operations utilizing geometric data.
The publication of D. Demandolx and J. Davoust, Multicolor Analysis and Local Image Correlation in Confocal Microscopy, Journal of Microscopy, Vol. 185 Pt. 1, January 1997, pp. 21–36 discloses a number of densitometric analysis methods. The individual analyses require both a geometric selection of the object to be analyzed and geometric selections in a specific analytical space (the cytofluorogram). The publication of P. Wedekind, U. Kubitschek, and R. Peters, Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging, journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 23–33, discloses a capability for overlaying onto an acquired image of an object geometric elements that are differently illuminated on the specimen, and effecting changes in the specimen by way of the energy transport associated therewith.
Standard microscope systems make available usually implemented in software geometric models suitable for this purpose (polygons, rectangles, more generally a “region of interest” or ROI) that the user defines. This usually requires a time-consuming interactive process. The region is drawn with the mouse on the display of a computer system; then it is made available to the corresponding automation function.